The present invention relates to the identification, isolation, sequencing and characterization of a new member of the histone deacetylase family, as well as its transcripts, gene products, associated sequence information, and related genes. The present invention also relates to methods for detecting and diagnosing carriers of normal and mutant alleles of these genes, methods for detecting and diagnosing diseases, methods of identifying genes and proteins related to or interacting with such genes and proteins, methods of screening for potential therapeutics for diseases, methods of treatment for diseases, and cell lines and animal models useful in screening for and evaluating potentially useful therapies for diseases. In a particular aspect of the present invention, a novel family member, HDAC7, is described, and its interaction with SMRT/N-CoR and mSin3A, its biochemical properties and subcellular localization are all characterized. In addition, evidence is provided that the HDAC4, 5, and 7 deacetylases mediate nuclear receptor repression. The findings described here indicate that two or more classes of hi stone deacetylases can collectively contribute to SMRT/N-CoR action and that at least some deacetylases may directly associate with SMRT/N-CoR in an mSin3A independent fashion.
Nuclear hormone receptors are sequence-specific and ligand-dependent transcription factors that control cell proliferation, differentiation, and animal physiology (Mangelsdorf and Evans, (1995) Cell, 83:841-850; Mangelsdorf et al., (1995) Cell, 83:835-839). They are structurally related and contain two evolutionarily conserved modules, the DNA binding (DBD) and ligand-binding domains (LBD). Several receptors including retinoic acid and thyroid hormone receptors function as potent repressors in the absence of ligands and as activators upon ligand binding. Intensive studies on the mechanism of transcriptional activation by nuclear hormone receptors led to the identification of coactivators including CBP/p300, PCAF, as well as the p160 family proteins (SRC-1; GRIP1/TIF2; ACTR/RAC3/P/CIP) (Blanco et al., (1998) Genes and Devel., 12:1638-51; Chen et al., (1997) Cell, 90:569-80; Hong et al., (1996) Proc. Natl. Acad. Sci. USA, 93:4948-52; Kamei et al., (996) Cell, 85: 403-414; Onate et al., (1995) Science, 270:1354-57; Torchia et al., (1997) Nature, 387:677-84; Yao et al., (1996) Proc. Nati. Acad. Sci. USA, 93:10626-31). Among these, CBP, PCAF, and SRC-1/ACTR have been recently shown to possess intrinsic histone acetyltransferase activity, consistent with a role for histone acetylation in transcriptional activation (Bannister and Kouzarides, (1996) Nature, 384:641-43; Chen et al., (1997) Cell, 90:569-80; Ogryzko et al., (1996) Cell, 87:953-59; Spencer et al., (1997) Nature, 389:194-98; Yang et al., (1996) Nature, 382:12845-50).
Several corepressors for nuclear receptors including SMRT, N-CoR, SUN-CoR, and Alien have also been identified (Chen and Evans, (1995) Nature, 377:454-57; Dressel et al., (1999) Cell. Blo., 19:3383-94; Horlein et al., (1995) Nature, 377:397-404; Ordentlich et al., (1999) Proc. Natl. Acad. Sci. USA, 96:2639-44; Zamir et al., (1996) Mol. Cell. Biol., 16:5458-65). SMRT and N-CoR were identified by yeast two-hybrid screens with nuclear receptors. Both proteins are large and possess at least four autonomous repression domains.
In addition to nuclear receptors, functional associations between SMRT/N-CoR with other transcription factors including CBF1/RBPJK, PLZF, BCL6, MyoD, Bach2, and Pbx1 have been demonstrated (Asahara et al., (1999) Mol. Cell. Biol., in press; Bailey et al., (1999) Mol. Endocrinol., 13:1155-68; Dhordain et al., (1997) Proc. Natl. Acad. Sci. USA, 94:10762-67; He et al., (1998) Nat Genet 18:126-35; Hong et al., (1997) Proc. Natl. Acad. Sci. USA, 94:9028-33; Huynh and Bardwell, (1998) Oncogene, 17:2473-84; Kao et al., (1998) Genes and Devel. 12:2269-77; Lin et al., (1998) Nature, 391:811-14; Muto et al., (1998) EMBO J., 17:5734-43; Wong and Privalsky, (1998) J. Biol. Chem., 273:27695-702), suggesting that corepressors, like coactivators, may function as signaling integrators to control cell fate. Several lines of evidence suggest that the mechanism underlying the repressive activity of SMRT and N-CoR corepressors is manifested through their recruitment of a histone deacetylase complex containing mSin3A and HDAC1 (Alland et al., (1997) Nature, 387:49-55; Hassig et al., (1997) Cell, 89:341-47; Heinzel et al., (1997) Nature, 387:43-48; Laherty et al., (1997) Cell, 89-349-56; Nagy et al., (1997) Cell, 89:373-80; Zhang et al., (1997) Cell, 357-64). Recruitment of acetylase/deacetylase complexes by coactivators/corepressors is thought to cause a local change in the chromatin structure, resulting in either activation or repression of gene transcription.
In yeast Sacchromyces cerevisiae, two distinct histone deacetylase complexes have been characterized (Carmen et al., (1996) J. Biol. Chem., 271:15837-44; Rundlett et al., (1996) Proc. Natl. Acad. Sci. USA, 93:14503-08). Histone deacetylase-B (HDB) is a 600 kDa complex which contains the Rpd3 protein. Histone deacetylase-A (HDA) is a 350 kDa complex and contains yeast Hda1 and the related Hos1, 2, and 3. Homology studies indicate that the HDA1-related deacetylases are structurally distinct from Rpd3 (which appears to be most related to mammalian class I deacetylases HDAC1, 2, and 3). Class II mammalian histone deacetylases (HDAC4, 5, and 6) have been recently identified which are structurally related to yeast Hda1 (Fischle et al., (1999) J. Biol. Chem., 274:11713-20; Grozinger et al., (1999) Proc. Natl, Acad. Sci. USA, 96:4868-73; Verdel and Khochbin, (1999) J. Biol. Chem., 2440-45). These family members are large in size (from 1085 amino acids to 1216 amino acids) and are able to deacetylate histones in vitro. HDAC4 and HDAC5 (also known as mHDA1) are highly homologous (51%/63% in identity/homology) and contain a conserved C-terminal deacetylase domain (89% amino acid identity). Intriguingly, HDAC6 (also known as mHDA2) has two catalytic domains at the amino-terminal, which have been suggested to form an intramolecular dimer. While HDAC4 has been shown to coprecipitate with HDAC3 and RbAp48, HDAC5 appears to associate with at least HDAC3 (Grozinger et al., 1999, supra). Furthermore, Northern blot analyses indicate that the tissue distribution patterns of family members are quite distinct. Numerous studies have indicated that the HDAC1/HDAC2 complexes are recruited to promoters by sequence-specific DNA-binding transcription factors (Doetzlhofer et al., (1999) Mol. Cell. Biol., 19:5504-11; Emiliani et al., (1998) Proc. Natl. Acad. Sci. USA, 95:2795-800; lavarone and Massague, (1999) Mol. Cell. Biol., 19:916-22; Radkov et al., (1999) J. Virol., 73:5688-97; Yang et al., (1996)). A recent report suggests that HDAC4 associates with and represses the MEF2 transcription factor (Miska et al., (1999) EMBO J., 18:5099-5107). However, the role of the HDAC4-6 family of histone deacetylases in transcription is largely unknown.
A key event in the regulation of eukaryotic gene expression is the posttranslational modification of nucleosomal histones, which converts regions of chromosomes into transcriptionally active or inactive chromatin. The most well studied posttranslational modification of histones is the acetylation of epsilon-amino groups on conserved lysine residues in the histones"" amino-terminal tail domains. Histone acetylation influences both gene transcription and chroma tin assembly after DNA replication and the enzymes that regulate this property of chromatin are likely to play a key role in regulating these crucial genomic functions. The steady-state level of histone acetylation is established and maintained by multiple histone acetyltransferases (HATs) and deacetylases (HDACs). Significant advances have been made in the past few years toward the identification of histone acetyltransferases and histone deacetylases.
The HDACs have been implicated in repression of gene expression by facilitating chromatin condensation and, like the HATs, operate as part of multi-protein complexes (Khochbin S, Wolffe AP FEBS Lett (1997) 419(2-3)157-60). More intriguing, at least some histone deacetylases are associated with chromatin-remodeling machines. The non-catalytic components of these complexes can either target the catalytic subunit to specific sites on the genome or regulate its enzymatic specificity. Kinase and phosphatase activities of intracellular signal transduction pathways may modify components of these complexes and thereby regulate their assembly, targeting or enzymatic function.
Recent efforts to understand the biological role of these enzymes reveals their involvement in cell-cycle regulation and differentiation. In addition, several studies have pointed to the possible involvement of histone deacetylases in human cancer. The availability of cloned histone deacetylase genes provides swift progress in the understanding of the mechanisms of deacetylases, their role in transcription, and their possible role in health and disease (Cress W D, Seto E., J Cell Physiol. 2000 Jul;184(1):1-16).
The present invention is based, in part, upon the identification, isolation, cloning and sequencing of a novel family member of the histone deacetylase superfamily. By the present invention, a novel family member, HDAC7, is described and its interaction with SMRT/N-CoR and mSin3A, its biochemical properties and subcellular localization are characterized. In addition, evidence is provided that the HDAC4, 5, and 7 deacetylases may mediate nuclear receptor repression.
Thus, in accordance with one embodiment of the present invention, there are provided isolated nucleic acids including nucleotide sequences comprising or derived from the HDAC7 genes and/or encoding polypeptides comprising or derived from the HDAC7 proteins.
In accordance with another embodiment of the present invention, there are provided host cells that have been transfected or otherwise transformed with one or more of invention nucleic acids.
In accordance with still another embodiment of the present invention, there are provided transgenic animal models for neoplasia and other diseases or disorders associated with mutations in HDAC7 genes.
In accordance with a further embodiment of the present invention, there are provided substantially pure protein preparations including polypeptides comprising or derived from HDAC7 proteins.
In accordance with another embodiment of the present invention, there are provided methods for the production and use of polyclonal and monoclonal antibodies, including antibody fragments, including Fab fragments, F(abxe2x80x2)2, and single chain antibody fragments, which selectively bind to HDAC7, or to specific antigenic determinants of HDAC7. The antibodies of the invention may be used in the various diagnostic, therapeutic and technical applications described herein.
In accordance with a still further embodiment of the present invention, there are provided methods of screening or identifying proteins, small molecules or other compounds which are capable of inducing or inhibiting the expression of HDAC7 genes and proteins.
In accordance with yet another embodiment of the present invention, there are provided methods for identifying proteins and other compounds which bind to, or otherwise directly interact with, HDAC7.
In accordance with still another embodiment of the present invention, there are provided methods of identifying proteins, small molecules and other compounds capable of modulating the activity of normal or mutant histone deacetylases, class I and/or class II, preferably HDAC7. In a particular aspect of the present invention, there are provided methods for identifying compounds capable of modulating specifically class I or class II histone deacetylases, more specifically, HDAC7.
In accordance with a further embodiment of the present invention, there are provided methods for screening for carriers of HDAC7 alleles associated with mutations in the HDAC7 genes. Screening and/or diagnosis can be accomplished, for example, by methods based upon the nucleic acids (including genomic and mRNA/cDNA sequences), proteins, and/or antibodies disclosed and enabled herein, including functional assays designed to detect failure or augmentation of the normal HDAC7 activity and/or the presence of specific new activities conferred by the mutant HDAC7s.
In yet another embodiment of the present invention, there are provided methods and pharmaceutical preparations for use in the treatment of histone deacetylase-associated diseases, e.g., HDAC7-associated diseases such as cancers, and the like.